This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Multidrug efflux pumps interfere significantly with cancer chemotherapy and the treatment of bacterial infections, by recognizing a number of structurally unrelated toxic compounds and actively extruding them from cells. Our long- term goal is to elucidate the structures and fundamental mechanisms that give rise to multiple drug recognition and extrusion in these multidrug transporters. The primary target of this proposal is the Escherichia coli AcrB transmembrane efflux pump, which shows the widest substrate specificity among all known multidrug transporters, ranging from most of the currently used antibiotics, disinfectants, dyes, detergents, to simple solvents. We have determined the x-ray structures of AcrB in the presence of four structurally different agents. These are the first structures of any transporter that have been solved in complex with a variety of ligands by x- ray crystallography. We continue to study the structure and function relationship of the AcrB efflux pump by x-ray crystallography and site-directed mutagenesis. We have generated and crystallized several AcrB mutants(D407A,D408A,K940A,and T978A. We will determine the x-ray structures of these AcrB mutants with a variety of structural dissimilar chemicals. AcrB exists as a complex with a periplasmic membrane fusion protein, AcrA. These two proteins presumably interact in the periplasm. The role of AcrA, which is absolutely required for transport, is still not known. To understand the mechanism of multidrug transport, the x- ray structure of the AcrAB complex is essential. We have co-crystallized the AcrB (native) and AcrA (with 6-his tag) efflux proteins. We will collect x- ray diffraction data of this AcrAB complex crystal. Visualizing the structure of this complex by crystallography will provide us direct us direct information about the mechanism of drug extrusion